Relative specificities of inhibition of acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase in cultured rabbit aortic smooth muscle cells by esterastin and cholesteryl oleyl ether

Rabbit aortic smooth muscle cells in culture were incubated with 0.04-500 M esterastin. Acid cholesteryl ester hydrolase (ACEH) and neutral cholesteryl ester hydrolase (NCEH) activities were inhibited to a comparable degree, with 50% inhibition occurring in the range of 0.4 M esterastin. Cells incubated with cholesteryl oleyl ether showed 50% inhibition of NCEH at 5.0 M, but no inhibition of ACEH over a concentration range of 0.2-20 M. This relative specificity of cholesteryl oleyl ether for NCEH can be employed to study the relative roles of ACEH vs. NCEH in preventing cellular cholesteryl ester accumulation.     

Cytotoxicity of cholesterol oxides and their effects on cholesterol metabolism in cultured human aortic smooth muscle cells

Incubation of monolayers of cultured human aortic smooth muscle cells with oxygenated sterols (25-hydroxycholesterol, 7-ketocholesterol, or cholesterol 5,6-epoxide) markedly inhibited growth though the viability of the culture was not affected. The effects on growth was concentration dependent, and 25-hydroxycholesterol was the most potent inhibitor of cellular growth as measured by decreased incorporation of thymidine into DNA and suppression of HMG-CoA reductase activity. The inhibitory effect of 25-hydroxycholesterol on cellular growth was not reversible if the cultures were grown in medium with normal fetal calf serum. However, in medium with delipidated serum, addition of purified cholesterol partially prevented growth inhibition induced by 25-hydroxycholesterol. Purified cholesterol, independently or in combination with tocopherol had no toxic effect on cellular growth. Addition of cholesterol oxides to the incubation medium stimulated lysosomal activation and release of acid phosphatase into the culture medium. The effect was concentration dependent and inversely related to cellular growth.     

Effect of phospholipids on the hydrolysis of cholesterol oleate liquid crystals by lysosomal acid lipase

Cholesterol oleate liquid crystals were prepared in vitro as a model of lipid droplets accumulated in the atheromatous aorta. The hydrolysis of cholesterol oleate crystals by lysosomal acid lipase was examined in the presence and absence of various phospholipids. Phosphatidylserine markedly stimulated the hydrolysis of cholesterol oleate liquid crystals (20 times the basal value); it increased the Vmax value about 15 times and decreased the Km value to 1/20 times the basal value. The polar head group and the acyl chains of phosphatidylserine were required for its stimulation of hydrolysis.     

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Inhibitory and stimulatory effects of phorbol ester on vasopressin-induced cellular responses in cultured rat aortic smooth muscle cells

In rat aortic smooth muscle cells, vasopressin (AVP) induces prostacyclin (PGI2) production, probably as the consequence of phospholipase C activation. Our study analyzes the effects of phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation on AVP-induced inositol 1,4,5-trisphosphate formation, cytosolic free Ca2+ concentration [( Ca2+]c), and PGI2 production. PMA rapidly decreased PKC activity in the cytosol of smooth muscle cells, while increasing it transiently in the membranes with a maximum around 20 min. Prior exposure of the cells to PMA resulted in a transient inhibition of both AVP-induced inositol 1,4,5-trisphosphate formation and [Ca2+]c rise. This was inversely correlated with membraneous PKC activity and partially reversed by the PKC inhibitor staurosporine. In contrast, pretreating the cells with PMA markedly potentiated A23187 or AVP-induced PGI2 production. Under those conditions, AVP-induced PGI2 production did not correlate either with PMA-induced membranous PKC activity or with AVP-induced PLC activation. However, this potentiating effect of PMA was reversed by staurosporine and was not mimicked by the 4 alpha-phorbol, an inactive analogue of PMA. Thus, the possibility is raised that, while inhibiting AVP-induced PLC activation, PMA-induced PKC activation increases the Ca2+ sensitivity of the cellular signaling system leading to PGI2 production.     

Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells

In addition to the well established cyclooxygenase pathway, cultured aortic smooth muscle cells convert arachidonic acid to several polar metabolites identified by high performance liquid chromatography and gaz chromatography — mass spectrometry. 15-Hydroxyeicosatetraenoic acid, 12-Hydroxyeicosatetraenoic acid and 5-Hydroxyeicosatetraenoic acid are the major products formed. These observations indicate that the rabbit aortic smooth muscle cells are a potential source of lipoxygenase products and raise the possibility that this pathway of arachidonic acid metabolism can influence the biological functions of arterial myocytes under normal and pathological conditions.     

Effect of anticalmodulin and antitubulin on LDL-receptor synthesis in aortic smooth muscle cells cultured in vitro

Effect of trifluoperazine and colchicine on LDL-receptor synthesis in smooth muscle cells exposed to hypercholesterolemic medium in vitro have been studied. While trifluoperazine at 25 microM concentration caused stimulation of LDL-receptor synthesis, colchicine acted as a dose-dependent inhibitor of LDL-receptor synthesis. Thus calmodulin down regulates LDL-receptor synthesis independent of microtubular involvement.     

Increase of PTP levels in vascular injury and in cultured aortic smooth muscle cells treated with specific growth factors

Migration and proliferation of vascular smooth muscle cells are key events in injury-induced neointima formation. Several growth factors and ANG II are thought to be involved in neointima formation. A recent report indicated that vascular injury is associated with increased mRNA levels of protein tyrosine phosphatase (PTP)-1B (PTP-1B). In the present study, we tested the following hypotheses: 1) rat carotid artery injury induces the expression of PTP-1B, Src homology-2 domain phosphatase (SHP-2), and PTP-proline, glutamate, serine, and threonine sequence (PEST) protein; and 2) polypeptide growth factors as well as ANG II increase the levels of tyrosine phosphatases in cultured rat aortic smooth muscle cells. We found that vascular injury induced by balloon catheter increases the protein levels of aforementioned phosphatases and that these effects occur in a PTP specific, as well as temporally and regionally specific, manner. Moreover, treatment of cultured primary rat aortic smooth muscle cells with PDGF or bFGF, but not with IGF1, EGF, or ANG II, increases PTP-1B, SHP-2, and PTP-PEST protein levels. These results suggest that increased PDGF and bFGF levels, occurring after vascular injury, may induce expression of several PTPs.     

Chondroitin sulfate-modified LDL induces increased cholesteryl ester synthesis and down-regulation of LDL receptors in smooth muscle cells and macrophages

Hypercholesterolemia induces increased transcytosis and accumulation of plasma lipoproteins in the arterial intima, where they interact with matrix proteins and become modified and reassembled lipoproteins. Chondroitin 6-sulfate-modified LDL (CS-mLDL) induces migration, proliferation, and lipid accumulation in human aortic smooth muscle cells (SMCs). To search for the mechanism(s) responsible for lipid accumulation, cultured SMC and macrophages were exposed to CS-mLDL, minimally modified LDL (mmLDL), and native LDL (as a control). Then the cellular uptake, degradation and expression of the LDL receptor (LDL-R) was determined using radioiodinated ligands, ACAT activity assay, fluorescence microscopy and RT-PCR. The uptake of CS-mLDL was 2-fold higher in SMC and 3-to 4-fold higher in macrophages as compared to LDL and mmLDL; the lysosomal degradation of CS-mLDL was slower in SMCs and considerably diminished in macrophages. Compared with LDL, CS-mLDL induced increased synthesis and accumulation of esterified cholesterol in SMCs (∼2-fold) and macrophages (∼10-fold) within an expanded acidic compartment. CS-mLDL and mmLDL down-regulate the gene expression of the LDL-R in the both cell types. Mechanisms of CS-mLDL-induced lipid accumulation in SMC and macrophages involve increased cellular uptake, and diminished cellular degradation that stimulates cholesterol ester synthesis and accumulation in cytoplasmic inclusions and in the lysosomal compartment in an undegraded form; modified lipoproteins induce down-regulation of LDL-R.     

青藤碱对家兔主动脉血管平滑肌细胞内游离钙浓度及蛋白激酶C的影响

观测青藤碱对培养家兔血管平滑肌细胞内游离钙浓度及正常和缺血缺氧刺激下蛋白激酶C（PKC）活性的影响。方法：Fura-2／AM作Ca^2＋指示剂,检测青藤碱对培养家兔主动脉血管平滑肌细胞静息Ca^2＋浓度及去甲肾上腺素,高K^＋,咖啡因刺激作用下的改变,并与钙拮抗剂维拉帕米进行对照研究;复制血管平滑肌细胞缺血缺氧模型,液闪仪测定PKC活性。结果：青藤碱剂量依赖性抑制高K^＋去极化引起[Ca^2＋]i升高,青藤碱10×10^-6mol.L^-1、3×10^-5mol.L^-1、10^-4mol.L^-1,对NE通过受体介导引起的[Ca^2＋]i增高也有明显抑制。但对静息状态下及咖啡因刺激的血管平滑肌细胞[Ca^2＋]i无明显影响。正常时,青藤碱处理后血管平滑肌细胞胞浆、胞膜PKC活性均升高;缺血缺氧状态下,胞浆PKC活性升高,但胞膜PKC活性降低,青藤碱处理后胞浆PKC活性下降,胞膜PKC活性上升。结论：青藤碱可能抑制血管平滑肌细胞电压依赖性钙通道和受体操纵性钙通道,降低细胞内游离钙水平。调节缺血缺氧条件下血管平滑肌细胞PKC活性。     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

Internalization and processing of human angiogenin by cultured aortic smooth muscle cells

Human angiogenin is a 14-kDa plasma protein with angiogenic and ribonucleolytic activities. Angiogenin binds specifically to aortic smooth muscle cells, activates second messenger pathways, and inhibits their proliferation. Human and bovine aortic smooth muscle cells were used to study the internalization and intracellular fate of human angiogenin at 37 degrees C. Using a specific antibody against angiogenin, we found that the internalized native protein was localized in the perinuclear region at 30 min and then dispersed throughout the cytoplasm. In conditions favoring receptor-mediated endocytosis, internalization of iodinated angiogenin showed a first peak at 5 min and then further increased for up to 24 h. The half-life of the molecule, calculated as 12 h in chase experiments, could contribute to its intracellular accumulation. In cell extracts, in addition to the 14-kDa protein, a 8.7-kDa fragment was observed at 24 h, and three fragments with molecular mass of 10.5, 8.7, and 6. 1 kDa were detected at 48 h. Our data point to a specific internalization and processing of human angiogenin by aortic smooth muscle cells.     

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.     

Effect of LDL concentration polarization on the uptake of LDL by human endothelial cells and smooth muscle cells co-cultured

To substantiate our hypothesis that concentration polarization of low-density Upoprotein （LDL） plays an important role in the localization of atherogenesis, we investigated the effects of wall shear stress and water filtration rate （or perfusion pressure） on the luminal surface LDL concentration （cw） and the LDL uptake by human vascular endothelial cells and smooth muscle cells co-cultured on a permeable membrane using a parallel-plate flow chamber technique and a flow cyto-metry method. The results indicated that the uptake of fluorescent labeled LDL （DiI-LDL） by the co-cultured cells was positively correlated with Cw in a non-linear fashion. When cw was low, the uptake increased very sharply with increasing Cw. Then the increase became gradual and the uptake was seemingly leveled out when Cw reached beyond 160 μg/ml. The present study therefore has provided further experimental evidence that concentration polarization may occur in the arterial system and have a positive correlation with the uptake of LDLs by the arterial wall, which gives support to our hypothesis regarding the localization of atherogenesis.     

Cell shape and arrangement of cultured aortic smooth muscle cells grown on collagen gels

Aortic smooth muscle cells (SMC) grown on conventional plastic culture dishes have morphological and functional properties of dedifferentiated cells in sub-culture. We examined the influence of collagen gels on the cell shape and arrangement. The cells grown on collagen gels showed a multilayered growth with formation of nodules. When the edge of the collagen gels was detached from the culture dish, the shape and arrangement of cells on the edge differed from that of the central, still attached region. The cells grown on floating collagen gels exhibited a spindle-like shape and were arranged in concentric circles. These findings suggest that the physical property of the substrate influences the cell shape and arrangement.     

Alpha-blocker, bunazosinhydrochloride decreases cytosolic Ca++ of cultured rat aortic smooth muscle cells

Effect of alpha-blocker, bunazosinhydrochloride on cytosolic Ca++ concentration of rat aortic smooth muscle cells (SMC) was studied. Marked and sustained decrease in cytosolic Ca++ concentration of SMC was observed following the addition of 10(-7) M bunazosinhydrochloride. Furthermore, 10(-7) M bunazosinhydrochloride completely blocked the phenylephrine induced increase in cytosolic Ca++ of rat aortic SMC. It is of interest that a decrease in cytosolic Ca++ of vascular SMC was caused by alpha-blocker.